The number of studies concerning silver nanoparticles (AgNPs) has increased, due in part to their potential uses for biomedical applications. These particles have been demonstrated in the elimination of the hepatitis B virus and the inhibition of the proliferation of various cancer cells
Nanomaterials have been widely studied over the past decades, due to their unique chemical and physical properties. At a nanoscale, the properties of materials depend significantly on their particle size and morphology. Silver nanoparticles (AgNPs) are one of the most common commercialized nanomaterials used in key biological and medical studies for applications such as antimicrobial agents, drug and gene delivery vehicles and biosensors. (
AgNPs have been demonstrated to effectively inhibit the replication of hepatitis B virus (HBV) (
In the clinic, conventional anti-fibrotic treatments and chemotherapy are of limited success in chronic liver injury, predominantly due to non-specific drug effects and the development of drug tolerance. Thus, AgNPs may provide a more efficacious and safer therapeutic approach. The possibility of this approach is important for hepatic stellate cells (HSCs), which are considered to be targets for the therapy of hepatic fibrosis and liver cirrhosis. Thus, investigation of the cytotoxicity of AgNPs in HSCs may be useful in determining their potential use in clinical applications.
In this study, the cytotoxicity of polyvinylpyrrolidone (PVP)-coated AgNPs in primary HSCs derived from fresh rat livers was determined. The biological responses of HSCs, such as the morphological changes in subcellular structure, proliferation, apoptosis, cell movement and cytokine secretion, were determined following the treatment of cells with AgNPs. Different particle sizes and concentrations of AgNPs were also used to study their effects on the cytotoxicity of AgNPs.
PVP-coated AgNPs with diameters of 10 and 30–50 nm were received in solution from Dr Jie Liu (Duke University, Durham, NC, USA). The size, morphology and dispersion of the nanoparticles were characterized using a Tecnai™ G2 Twin Transmission Electron Microscope (FEI, Hillsboro, OR, USA) and dynamic light scattering (Compact Goniometer System 3; ALV-GmbH, Langen, Germany). The ζ potential was determined using a Zetasizer Nano ZS (Malvern Instruments, Malrem, UK). X-ray diffraction experiments were performed on powder samples and were analyzed using an X’Pert PRO MRD HR diffractometer with a Cu Kα radiation (1.5405 Å) at 5 kV and 40 mA (PANalytical, Almelo, Netherlands). X-ray photoelectron spectroscopy (Kratos Analytical Inc., Chestnut Ridge, Spring Valley, NY, USA) was used to determine the composition of the surface coating.
HSCs were isolated from the livers of six normal Buffalo rats aquired from the Chinese Academy of Sciences (Shanghai, China) using the improved Friedman method (
Transmission electron microscopy (TEM; CM12; Philips, Amsterdam, Netherlands) was used to examine the morphology of HSCs. HSCs were seeded in 100-mm tissue culture dishes, cultured for 2 days and treated with different concentrations of AgNPs for 24 h.
Cell proliferation was evaluated with a Cell Counting Kit-8 (CCK-8; Becton Dickinson). Approximately 5×103 cells were plated in each well of a 96-well plate. The cells were divided into seven groups. Group 1 served as a blank control; groups 2, 3, and 4 were treated with 30–50 nm AgNPs at 20, 100 and 250 μg/ml, respectively; and groups 5, 6 and 7 were treated with 10 nm AgNPs at 20, 100 and 250 μg/ml, respectively. Following incubation for 96 h, 10 μl WST-8 was added to the wells and the absorbance at 450 nm was determined using a microplate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The aforementioned samples were analyzed for 24 h.
The FITC-Annexin V and propidium iodide (PI) double staining method was used to detect apoptosis induced by AgNPs. Freshly isolated HSCs were cultured in 96-well plates for 2 days and were divided into seven groups as described previously for the cytotoxicity studies. Following 24 h of nanoparticle treatment, the cells were washed with cold phosphate-buffered saline (4°C) and stained using the FITC-Annexin V Apoptosis Detection kit (ExCell Biology, Inc., Shanghai, China). The stained cells were analyzed by flow cytometry by a trained laboratory technician according to the experimental protocol.
Rat LDH kits (Sigma-Aldrich) were used to conduct the LDH leakage assay. Two days following seeding in 24-well plates, the cells were divided into two groups. One group was treated with AgNPs of different diameters and different concentrations, and the other group was treated as a control without nanoparticle treatment. The two groups were incubated at 37°C with 5% CO2 for 4 h according to the manufacturer’s instructions. The cell culture medium (50 μl) was collected from each well and analyzed using a spectrometer (UV-Vis-NIR spectrophotometer; Agilent, Santa Clara, CA, USA).
The cell biological behaviors, including the total cell number, number of dead cells and cell movement, were measured using a real-time cell-monitoring system with a cell-culturing platform (Cell-IQ; Chip-Man Technologies, Tampere, Finland). HSCs were cultured in the Cell-IQ system in 24-well plates (1×104 cells/well) for 72 h. Cells were divided into five groups with two wells per group. One group served as a blank control and the remaining four groups were treated with large and small AgNPs at 20 and 100 μg/ml, respectively.
The HSCs were adjusted to a concentration of 5×105 cells/ml and cultured in 24-well plates. The 24 wells were divided into three groups; one group served as the blank control and the other two groups were treated with AgNPs (with diameters of either 10 or 30–50 nm) at a concentration of 20 μg/ml. The conditioned medium was collected after 2 days and the quantity of hepatocyte growth factor (HGF), interleukin (IL)-6, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, matrix metallopeptidase (MMP)-2 and MMP-9 in the serum-free HSC-conditioned medium was quantified using an enzyme-linked immunosorbent assay (ELISA) kit (ExCell Biology, Inc.) according to the manufacturer’s instructions.
All experiments were repeated at least three times, and the data are presented as the mean ± standard deviation. Student’s t-test was performed to determine the statistical significance of the difference between untreated cells (blank control) and cells treated with AgNPs. P<0.05 was considered to indicate a statistically significant difference.
TEM images of the AgNPs used in this study are shown in
Approximately 2×107 HSCs were isolated from each rat. The morphology of freshly isolated HSCs exhibited no obvious features (
According to the TEM analysis, AgNPs were rapidly internalized by HSCs, although the majority of the nanoparticles were observed on the cell surface and between the cells (
To investigate the mechanism of cell death induced by AgNPs, the treated cells were stained with FITC-Annexin V and PI. The smaller AgNPs exhibited a greater ability to induce apoptosis and necrosis in the HSCs than the larger AgNPs (
The effect of AgNPs on the plasma membrane was not statistically significant at any of the concentrations tested (P>0.05;
The time-dependent cytotoxicity of AgNPs was assessed using the CCK-8 assay (
Dynamic alterations of the total cell number, number of dead cells and cell movement were measured using a Cell-IQ culturing platform. The increased percentage of the total cell number was significantly lower following incubation with AgNPs, than in untreated cells (P<0.05). The inhibitory effects of AgNPs were dependent on the size and dose (
Analysis of cytokine levels following treatment of HSCs with AgNPs was performed using ELISA. Production of HGF, IL-6, TGF-β1, and TNF-α was not significantly different between the treated and untreated groups (
Previously, AgNPs have been studied as a result of their ability to inhibit HBV and their function in drug delivery and targeting, which may lead to their application for treating liver diseases (
Previous studies have demonstrated the impact of nanoparticle size on cellular uptake and consequent cytotoxicity (
The changes in HSC morphology were studied using phase contrast microscopy and TEM prior to and following incubation with two sizes of AgNPs at various concentrations. These studies indicated that AgNPs induced HSCs apoptosis or necrosis. The ultrastructural characteristics of apoptosis are cell shrinkage, karyopyknosis, karyorrhexis and karyolysis. Karyorrhexis is a type of destructive fragmentation of the nucleus and is preceded by pyknosis and followed by karyolysis (
The mitochondrial swelling observed following incubation of HSCs with AgNPs indicated that the AgNPs in the cytoplasm primarily reside in the mitochondria, affecting their function and consequently exerting effects on cell metabolism. Mitochondria are important signaling centers during apoptosis, and the loss of mitochondrial integrity is induced and inhibited by numerous regulators of apoptosis (
Activated HSCs are important in chronic liver disease through the production of various cytokines. The ELISA assay confirmed that AgNPs inhibited the production of MMP-2 and −9, which are known to be crucial in chronic liver injury. During hepatic inflammation and hepatocellular necrosis, MMP-2 and −9 are predominantly produced by myofibroblasts, which transdifferentiate from activated HSCs (
In conclusion, the cytotoxicity of AgNPs was investigated by various biochemical approaches and was particle size- and dose-dependent. Morphology alterations may be an indication of metabolic and structural disturbances of HSCs caused by AgNPs. Thus, it was concluded that these alterations were size-dependent, as smaller particles induced greater cellular damage at the same concentrations. The results of the LDH assay confirmed that the necrosis or apoptosis of HSCs was not due to the acute toxicity of AgNPs but due to the effects of the nanoparticles on the physiological activities of the cells. These findings may illustrate the relative safety of AgNP application to the human body. AgNPs also affected the HSC cytokine secretion, as demonstrated by the inhibitory effects of AgNPs on the production of MMP-2 and −9. These results suggested that AgNPs may be used in the treatment of hepatic fibrosis, including HCC; however, the molecular mechanisms of nanoparticle cytotoxicity remain unclear. Therefore, further studies are required to elucidate particle-cell interactions and the metabolic and immunological responses activated in HSCs in the presence of AgNPs.
This study was conducted at Duke University and was supported by the National Science Foundation (NSF) and the Environmental Protection Agency (EPA) under NSF Cooperative Agreement EF-0830093, Center for the Environmental Implications of NanoTechnology. Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the NSF or the EPA. This study has not been subjected to EPA review and no official endorsement should be inferred.
Characterization of silver nanoparticles (AgNPs). (Aa) Transmission electron microscopy (TEM) images for small AgNPs. The mean diameter was 30±10 nm. (Ab) TEM images for large AgNPs. The mean diameter was 80±40 nm. (B) Ultraviolet-visible (UV-Vis) absorption spectra of both AgNPs were acquired with a Cary 500 scan UV-Vis-NIR spectrophotometer. The material was polydispersed, and the broad absorption >500 nm indicated the possible existence of large aggregates. (C) The characteristic X-ray diffraction pattern of the two sizes of AgNPs used was obtained using a X’Pert PRO MRD HR diffractometer. The AgNPs appeared as metallic silver with a face-centered cubic lattice. (D) Axis Ultra X-ray photoelectron spectroscopy was used to evaluate the elemental composition in the two sizes of nanoparticles with respect to C, O, N and Ag, using anode mode and aluminum monochromatic energy sources.
Characterization of hepatic stellate cells (HSCs) isolated from rat livers. To identify the HSCs, the newly isolated cells were cultured for 2 days (quiescent HSCs), 4 days and 7 days (activated HSCs). (A) Giemsa staining for the quiescent HSCs. The typical light microscopic appearance of lipid droplets is shown. (B) Fluorescence microscope image depicts the cyan vitamin A auto-fluorescence of quiescent HSCs. (C) Following culture for 4 days, HSCs exhibited a distinct star-like morphology in optical micrographs. (D) Activated HSCs demonstrated fibroblast-like morphology in optical micrographs.(E) Immunofluorescence analysis of desmin expression in qHSCs. Immunofluorescence staining for desmin was performed using an anti-desmin antibody.(G) α-smooth muscle actin (SMA)-positive activated HSCs. Immunofluorescence staining for α-SMA was performed using an anti-α-SMA antibody. (F) Desmin and (H) α-SMA staining for HSC nuclei.
The morphology of hepatic stellate cells (HSCs) incubated with varying concentrations of two different sizes of silver nanoparticles (AgNPs; arrows) indicated that apoptosis or necrosis was induced by the AgNPs. (A) Optical micrographs of HSCs treated with smaller AgNPs at 100 μg/ml for 24 h. AgNPs accumulated on the cell surface and between cells. (B) Transmission electron microscopy (TEM) images of HSCs treated with larger AgNPs at 100 μg/ml for 24 h demonstrated that the AgNPs aggregated in the cytoplasm. (C) HSCs incubated with 250 μg/ml of small nanoparticles for 7 days exhibited karyorrhexis, karyolysis and karyotheca disintegration in TEM images. (D) HSCs treated with 20 μg/ml of large nanoparticles for 2 days exhibited mitochondrial swelling by TEM. (E and F) HSCs without any treatment exhibited a normal cell structure under TEM.
Inhibitory effects of silver nanoparticles (AgNPs) on the bio-behaviors of hepatic stellate cells (HSCs). Following culture of HSCs with or without AgNPs of the two sizes at either 20 μg/ml or 100 μg/ml for 72 h, the targets were measured by a real-time cell-monitoring system. Images were captured at 5-min intervals for 12 h. Data are presented as the mean ± SD, and each group comprises eight measurements. (Aa) Control cells (untreated). (Ab) Cells treated with large AgNPs at 100 μg/ml. (Ac) Cells treated with small AgNPs at 100 μg/ml. (Ba) Dead cells as a proportion (%) of total cells were calculated according to the following formula: Rate (%) = number of dead cells/number of primary seeded cells × 100. (Bb) Cell movement was calculated as the sum of the distance of movement of each cell between two images (5-min intervals)/number of primary seeded cells. (Bc) Increased percentage (%) of total cell number was calculated as the following: Rate (%) = (value at each time point-value of primary seeding cells)/value of primary seeding cells ×100.
Influence of silver nanoparticles (AgNPs) on cytokine production. Following incubation of hepatic stellate cells (HSCs) with AgNPs of different sizes at 0.2 mg/ml for 48 h, the levels of various cytokines [hepatocyte growth factor (HGF), interleukin (IL)-6, transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, matrix metallopeptidase (MMP-2) and MMP-9] in the medium were measured using an enzyme-linked immunosorbent assay kit. Statistical analysis showed that the levels of MMP-2 and −9 were significantly different in cultures of cells incubated with AgNPs as compared with the medium from the untreated cells (P<0.05), while the other four groups were not significantly different compared with the control group (P>0.05). Control, HSCs treated without AgNPs; large sized, HSCs treated with AgNPs, with a diameter of 30–50 nm; small sized, HSCs treated with AgNPs, with a diameter of 10 nm.
Characterization of silver nanoparticles.
Silver nanoparticle | ||
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| ||
Property | Small | Large |
Nominal diameter (nm) | 10 | 30–50 |
TEM diameter (nm) | 30±10 | 80±40 |
Hydrodynamic radius using DLS (nm) | 28.6±0.61 (PDI: 0.395) | 58.74±1.49 (PDI: 0.491) |
ζ Potential (mV) | −24.5±17.2 | −28.6±5.54 |
Silver recovery using ICP-MS (%) | 85.2 | 87.3 |
Data are presented as the mean ± SD. TEM, transmission electron microscopy; DLS, dynamic light scattering; PDI, polydispersity index; ICP-MS, inductively coupled plasma mass spectrometer (Agilent, Santa Clara, CA, USA).